siRNA And Their Use In Methods And Compositions For Inhibiting The Expression Of The ORAI1 Gene

ABSTRACT

The invention relates to siRNA molecules and their use in methods and pharmaceutical compositions for inhibiting the expression of the ORAil gene. The invention also relates to the use of said siRNAs molecules in the treatment and/or prevention of an eye condition characterised by increased expression and/or activity of ORAil gene, preferably said eye condition is conjunctivitis and/or an ocular allergy such as seasonal allergic conjunctivitis, perennial allergic conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, and giant papillary conjunctivitis.

FIELD OF THE INVENTION

The present invention relates to the field of siRNA products and theiruse in methods and compositions for the treatment and/or prevention ofeye conditions, and more particularly for the treatment and/orprevention of eye conditions such as conjunctivitis and/or ocularallergy, related to high levels of expression and or activity of ORAI1.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a naturally occurring post-transcriptionalregulatory mechanism present in most eukaryotic cells that uses smalldouble stranded RNA (dsRNA) molecules to direct homology-dependent genesilencing. Its discovery by Fire and Mello in the worm C. elegans {Fire,1998} was awarded the Nobel Prize in 2006. Shortly after its firstdescription, RNAi was also shown to occur in mammalian cells, notthrough long dsRNAs but by means of double-stranded small interferingRNAs (siRNAs) 21 nucleotides long {Elbashir, 2001}.

The process of RNA interference is thought to be anevolutionarily-conserved cellular defence mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse phyla andflora, where it is called post-transcriptional gene silencing. Since thediscovery of the RNAi mechanism there has been an explosion of researchto uncover new compounds that can selectively alter gene expression as anew way to treat human disease by addressing targets that are otherwise“undruggable” with traditional pharmaceutical approaches involving smallmolecules or proteins.

According to current knowledge, the mechanism of RNAi is initiated whenlong double stranded RNAs are processed by an RNase III-like proteinknown as Dicer. The protein Dicer typically contains an N-terminal RNAhelicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ)domain, two RNase III domains and a double-stranded RNA binding domain(dsRBD) {Collins, 2005} and its activity leads to the processing of thelong double stranded RNAs into 21-24 nucleotide double stranded siRNAswith 2 base 3′ overhangs and a 5′ phosphate and 3′ hydroxyl group. Theresulting siRNA duplexes are then incorporated into the effector complexknown as RNA-induced silencing complex (RISC), where the antisense orguide strand of the siRNA guides RISC to recognize and cleave targetmRNA sequences {Elbashir, 2001} upon adenosine-triphosphate(ATP)-dependent unwinding of the double-stranded siRNA molecule throughan RNA helicase activity {Nykanen, 2001}. The catalytic activity ofRISC, which leads to mRNA degradation, is mediated by the endonucleaseArgonaute 2 (AGO2) {Liu, 2004; Song, 2004}. AGO2 belongs to the highlyconserved Argonaute family of proteins. Argonaute proteins are −100 KDahighly basic proteins that contain two common domains, namely PIWI andPAZ domains {Cerutti, 2000}. The PIWI domain is crucial for theinteraction with Dicer and contains the nuclease activity responsiblefor the cleavage of mRNAs {Song, 2004}. AGO2 uses one strand of thesiRNA duplex as a guide to find messenger RNAs containing complementarysequences and cleaves the phosphodiester backbone between bases 10 and11 relative to the guide strand's 5′ end {Elbashir, 2001}. An importantstep during the activation of RISC is the cleavage of the sense orpassenger strand by AGO2, removing this strand from the complex {Rand,2005}. Crystallography studies analyzing the interaction between thesiRNA guide strand and the PIWI domain reveal that it is onlynucleotides 2 to 8 that constitute a “seed sequence” that directs targetmRNA recognition by RISC, and that a mismatch of a single nucleotide inthis sequence may drastically affect silencing capability of themolecule {Ma, 2005; Doench 2004; Lewis, 2003}. Once the mRNA has beencleaved, due to the presence of unprotected RNA ends in the fragmentsthe mRNA is further cleaved and degraded by intracellular nucleases andwill no longer be translated into proteins {Orban, 2005} while RISC willbe recycled for subsequent rounds {Hutvagner, 2002}. This constitutes acatalytic process leading to the selective reduction of specific mRNAmolecules and the corresponding proteins. It is possible to exploit thisnative mechanism for gene silencing with the purpose of regulating anygene(s) of choice by directly delivering siRNA effectors into the cellsor tissues, where they will activate RISC and produce a potent andspecific silencing of the targeted mRNA. RNAi has been applied inbiomedical research such as treatment for HIV, viral hepatitis,cardiovascular and cerebrovascular diseases, metabolic disease,neurodegenerative disorders and cancer {Angaji SA et al 2010}.

Many studies have been published describing the ideal features a siRNAshould have to achieve maximum effectiveness, regarding length,structure, chemical composition, and sequence. Initial parameters forsiRNA design were set out by Tuschl and co-workers in WO02/44321,although many subsequent studies, algorithms and/or improvements havebeen published since then. siRNA selection approaches have become moresophisticated as mechanistic details have emerged, in addition furtheranalysis of existing and new data can provide additional insights intofurther refinement of these approaches {Walton SP et al 2010}.Alternatively, several recent studies reported the design and analysisof novel RNAi-triggering structures distinct from the classical 19+2siRNA structure and which do not conform to the key features ofclassical siRNA in terms of overhang, length, or symmetry, discussingthe flexibility of the RNAi machinery in mammalian cells {Chang CI et al2011}.

Also, a lot of effort has been put into enhancing siRNA stability asthis is perceived as one of the main obstacles for therapy based onsiRNA, given the ubiquitous nature of RNAses in biological fluids.Another inherent problem of siRNA molecules is their immunogenicity,whereby siRNAs have been found to induce unspecific activation of theinnate immune system. The knockdown of unintended genes (mRNAs) is awell-known side effect of siRNA-mediated gene silencing. It is caused asa result of partial complementarity between the siRNA and mRNAs otherthan the intended target and causes off-target effects (OTEs) from geneshaving sequence complementarity to either siRNA strand. One of the mainstrategies followed for stability enhancement and OTE reduction has beenthe use of modified nucleotides such as 2′-O-methyl nucleotides,2′-amino nucleotides, or nucleotides containing 2′—O or 4′—C methylenebridges. Also, the modification of the ribonucleotide backboneconnecting adjacent nucleotides has been described, mainly by theintroduction of phosphorothioate modified nucleotides. It seems thatenhanced stability and/or reduction of immunogenicity are ofteninversely proportional to efficacy {Parrish, 2000}, and only a certainnumber, positions and/or combinations of modified nucleotides may resultin a stable and non-immunogenic silencing compound. As this is animportant hurdle for siRNA-based treatments, different studies have beenpublished which describe certain modification patterns showing goodresults, examples of such include EP1527176, WO2008/050329,WO2008/104978 or WO2009/044392, although many more may be found in theliterature {Sanghvi YS. 2011; Deleavey et al 2012}.

Allergic diseases are characterized by an overreaction of the humanimmune system to a foreign protein substance (“allergen”) that is eaten,breathed into the lungs, injected or touched. Allergies have a geneticcomponent. If only one parent has allergies of any type, chances are 1in 3 that each child will have an allergy. If both parents haveallergies, it is much more likely (7 in 10) that their children willhave allergies. There are no cures for allergies; however they can bemanaged with proper prevention and treatment.

About 30% of people worldwide suffer from allergic symptoms and 40-80%of them have symptoms in the eyes {Key B. 2001}. Allergic diseasesaffecting the eyes or ocular allergies constitute a heterogenic group ofdiseases with a very broad spectrum of clinical manifestations. Anocular allergy usually occurs when the conjunctiva (membrane coveringthe eye and the lining of the eyelid) reacts to an allergen. An ocularallergy can happen independently or in conjunction with other allergysymptoms (such as rhinitis or asthma).

Basic and clinical research has provided a better understanding of thecells, mediators, and immunologic events which occur in ocular allergy.The eye, particularly the conjunctiva, has a relatively large number ofmast cells. When allergens are present they can bind to immunoglobulin,IgE, in the FcξRI receptors on the surface of these mast cells andtrigger their activation and release of mediators of allergy (a processknown as degranulation). Degranulation releases mast cell components,including histamine, prostaglandins, tryptase and leukotrienes, into theenvironment outside the mast cell. Through a variety of mechanisms thesecomponents produce the signs and symptoms of the ocular allergy. Theactivation of the mast cells of the allergic inflammation is frequentlydesignated as an acute phase response or early phase of the ocularallergy. The acute phase response can progress to a late phase responsecharacterized by recruitment of inflammatory cells to the site of theallergic inflammation, for example as an influx of eosinophils andneutrophils into the conjunctiva.

Ocular allergy represents one of the most common conditions encounteredby allergists and ophthalmologists. Ocular allergy is usually associatedwith the following symptoms and signs: conjunctivitis, blepharitis,blepharoconjunctivitis or keratoconjunctivitis. The eye becomes red,itchy and there occurs lacrimation and slight discharge. Severe casesmay also show eye burning sensation, pain and photophobia.

Allergic diseases affecting the eyes include mild forms such as seasonalallergic conjunctivitis (SAC) and perennial allergic conjunctivitis(PAC); and more severe manifestations such as vernalkeratoconjunctivitis (VKC); atopic keratoconjunctivitis (AKC) and giantpapillary conjunctivitis (GPC). The latter ones can be associated withcomplications such as corneal damage and may cause vision loss. SAC andPAC are commonly IgE-mast cell mediated hypersensitivity reaction toexternal allergens; whereas AKC and VKC are characterized by chronicinflammation involving several immune cell types. SAC and PAC allergens,with the help of antigen presenting cells (APCs), trigger aTh2-predominant immune response that induces B cells to release IgE.Activation of the allergic response usually involves infiltration anddegranulation of mast cells.

SAC is the most common allergic disease in the eye, usually caused byallergens like airborne pollen, dust, and animal dander. The signs andsymptoms usually occur during the spring and summer, and generally abateduring the winter months. Itching, redness and swelling of theconjunctiva are the most characteristic symptoms, but also tearing,burning sensation, and photophobia. In most cases, SAC is not serious.However, it may be very disturbing to patients because it can affecttheir quality of life and can have significant socioeconomic impact{Kari O and Saari KM 2010}.

PAC is the second most common allergic disease in the eye, usuallycaused by animals and mites. The symptoms and signs are much the same asin SAC, the difference is the specific allergens to which the patient isallergic and that PAC can occur throughout the year with exposure toperennial allergens. PAC affects all age groups but mostly young andmiddle-aged people of both sexes. In addition, PAC is often connected todry eye syndrome.

SAC and PAC are the most common forms of ocular allergies. Estimatesvary, but these types of allergy are said to affect at least 15-20% ofthe general population. SAC and PAC are often underdiagnosed andconsequently undertreated. In SAC and PAC allergen induced local releaseof IgE prompts degranulation of mast cells in Ca2+ dependent mechanism.IgE-activated mast cells liberate preformed inflammatory mediators suchas histamine and leukotriene 4 that are the first mediators of theallergic response. These mediators attract eosinophils that infiltratethe region amplifying the allergic response.

VKC is a relatively rare chronic allergic inflammation of the ocularsurface that mainly affects children and young adolescents. Mainsymptoms are itching, redness, swelling, discharge and photophobia. Themost characteristic sign is giant papillae in the upper tarsalconjunctiva.

AKC is a bilateral chronic inflammatory disease of the ocular surfaceand eyelid. The most characteristic sign are eczematous lesions on theeyelid which are itchy. It is not unusual for AKC patients to havecataract surgery at a young age {Kari O and Saari KM 2010}.

GPC is an inflammatory disease characterized by papillary hypertrophy ofthe superior tarsal conjunctiva. GPC is caused by inert substancesrather than allergens. When these irritative stimuli are removed theconjunctival papillary changes resolve. Protein deposits on the surfaceof the contact lens could become antigenic and stimulate the productionof IgE {La Rosa M. et al 2013}.

Current treatments for ocular allergy include non-pharmacologic andpharmacologic strategies. Avoidance of the antigen is the primarybehavioural modification for all types of ocular allergies. Artificialtear substitutes provide a barrier function and help to improve thefirst-line defence at the level of the conjunctiva mucosa. Whennon-pharmacologic strategies do not provide adequate symptom relief,pharmacologic treatments may be applied.

The mainstay of the management of ocular allergy involves the use ofanti-allergic therapeutic agents such as antihistamine, dual-action orcombination treatments and mast cell stabilizers. Topical antihistamines(such as Emedastine and Levocabastine) competitively and reversiblyblock histamine receptors and relieve itching and redness, but only fora short time. Antihistamines do not affect other proinflammatorymediators which remain inhibited. A limited duration of actionnecessitates frequent dosing and topical antihistamines may beirritating to the eye, especially with prolonged use.

Combination treatments using decongestants (such as oxymetazoline,tetrahydrozoline, and naphazonline) in combination with antihistaminesact as vasoconstrictors but are known to sting or burn on instillation.Other adverse events include mydriasis and rebound hyperemia, renderingthese combination treatments more suitable for short-term relief. Inaddition, these drugs are not recommended for use in patients withnarrow-angle glaucoma. Mast cell stabilizers (such as cromoglycate,lodoxamide, nedocromil) have a mechanism of action that is unclear. Theydo not relieve existing symptoms and can be used only on a prophylacticbasis to prevent mast cell degranulation with subsequent exposure to theallergen. They require a loading period during which they must beapplied before the antigen exposure {La Rosa M. et al 2013}.

When the above mentioned anti-allergic drugs do not allow adequatecontrol of the allergic inflammatory process, anti-inflammatory agentsare used. Corticosteroids remain among the most potent pharmacologicagents used in the more severe variants of ocular allergy {La Rosa M. etal 2013}. However, steroidal drugs can have side effects that threatenthe overall health of the patient. Chronic administration ofcorticosteroids can lead to drug-induced osteoporosis by suppressingintestinal calcium absorption and inhibiting bone formation. Otheradverse side effects of chronic administration of corticosteroidsinclude hypertension, hyperglycemia, hyperlipidemia (increased levels oftriglycerides) and hypercholesterolemia (increased levels ofcholesterol) because of the effects of these drugs on the body metabolicprocesses. It is also known that certain corticosteroids have a greaterpotential for elevating intraocular pressure (“IOP”) than othercompounds in this class. For example, it is known that prednisolone,which is a very potent ocular anti-inflammatory agent, has a greatertendency to elevate IOP than fluorometholone, which has moderate ocularanti-inflammatory activity. It is also known that the risk of IOPelevations associated with the topical ophthalmic use of corticosteroidsincreases over time. In other words, the chronic (i.e., long-term) useof these agents increases the risk of significant IOP elevations.Therefore, corticosteroids may not be appropriate for the long-termtreatment of ocular allergies. In addition, chronic use ofcorticosteroids is contraindicated due to an increased risk for thedevelopment of cataracts and glaucoma {Ono SJ, and Abelson MB, 2005}.

Allergy immunotherapy is useful in reducing the response to allergens,but its role in allergic conjunctivitis has not been proven. The mainobjective of this treatment is to induce clinical tolerance to thespecific allergen. The therapy is administered subcutaneously inprogressively increasing doses to remain below the threshold of aclinical reaction. Sublingual immunotherapy (SLIT) is considered analternative to subcutaneous allergy immunotherapy and is administeredorally under the tongue, but long-term results with SLIT are not yetavailable. Most of the trials with this form of therapy have been forallergic rhinitis. In general, immune responses to allergenadministration are not predictive of the effectiveness of the therapyand the therapy itself can produce systemic reactions, the incidence andseverity of which vary dependent of the type of allergen administered{La Rosa M. et al 2013}.

In addition, the majority of newer ophthalmic anti-allergic agents havelimited durations of action and twice daily dosing is required. Atopical preparation with a longer duration of action would beadvantageous because it may be instilled once daily. Thus, new therapiesthat can offer advantages in areas such as efficacy and duration ofaction, while offering similar safety profiles than traditionalophthalmic anti-allergic agents, are needed.

RNA interference-based therapies have been pointed out as having thepotential to satisfy unmet needs in allergy treatment {Popescu FD.2005}. It has been demonstrated that systemic administration of CD40siRNA in mice sensitized with an allergen is capable of attenuatingnasal allergic symptoms through inhibition of dendritic cell and B cellfunctions and generation of regulatory T cells {Suzuki M. et al 2009}.In addition, siRNA-based allergen-specific therapy for allergic rhinitishas also been developed by using CD40-silenced and allergen-pulseddendritic cells {Suzuki M et al 2010}.

Changes in the intracellular free calcium (Ca2+) concentration in thecell regulate many biological cell functions. Ca2+ signals are generatedby the controlled release of Ca2+ from the endoplasmic reticulum (ER)which is the major calcium store in cells. Calcium release from the ERactivates store-operated Ca2+(SOC) channels at the plasma membrane,triggering the store-operated Ca2+ entry (SOCE) of extracellular calciuminflux into the cytoplasm. Recent studies highlighted the importance ofthis Ca2+ entry mechanism in a variety of pathophysiological processes,including allergy {Bergmeier W et al 2013}.

SOCE is activated in response to depletion of ER Ca2+ pools. Activationof SOCE induces Ca2+ entry from extracellular compartments and this ismediated by store-operated Ca2+ release-activated Ca2+ (CRAC) channels{Hoth M. et al 1992}.

CRAC channels are composed of calcium sensing proteins called STIM(stromal interaction molecule) and pore-forming subunits named ORAI.Mammalian cells have three ORAI isoforms: ORAI1, ORAI2 and ORAI3;although ORAI2 and ORAI3 fulfill the same role as ORAI1 the Ca2+currents generated by these proteins are around two- to three foldsmaller than the ones generated by ORAI1 {Smyth J.T. et al 2006}.

ORAI1 is a widely expressed, 33 kDa plasma membrane protein with 4transmembrane domains that is activated by the calcium sensor stromalinteraction molecule 1 (STIM1) when calcium stores are depleted andinduces extracellular calcium influx into the cytoplasm through the CRACchannels. ORAI1 is also called CRAC Modulator 1, CRACM1, ORAT1,Transmembrane Protein 142A, TMEM142A, or CRAC Channel Protein 1. ORAI1has been defined as the key subunit of the CRAC channels {Wang Y. et al2012}.

There is growing evidence that indicates that short-term and long-termactivation of immune cells in allergic responses is mediated by influxof Ca2+ to immune cells from the extracellular compartment. Short-termresponses include degranulation of mast cells and activation of effectorcytolytic T cells. Indeed, mast cells lacking either STIM1 or ORAI1 showa considerable defect in degranulation {Vig M. et al 2008; Baba Y. et al2008}. Long-term responses involve modulation of gene expression thatcontrols B and T cell proliferation and differentiation {Parekh A.B. etal 2005}.

It has also been suggested that ORAI1 is crucial in mouse mast celleffector function. Mast cells derived from ORAI1-deficient mice showedgrossly defective degranulation and the induction of the IgE mediated invivo passive anaphylaxis response is also dependent on ORAI1. {Vig M, etal 2008}.

ORAI1 knockdown by RNAi has been studied in allergic rhinitis.Recombinant lentivirus vectors that encoded shRNA (short hairpin RNA)against ORAI1 administered into the nostrils of OVA-sensitized micealleviated allergic rhinitis symptoms (number of sneezes and number ofnasal rubbings) {Wang et al 2012}. Rhinitis is produced by nasalirritation or inflammation due to blockage or congestion. Allergicrhinitis is allergic inflammation in the upper airway associated withhyperresponsiveness of several types of immune cells {Wang et al 2012}.However not all the rhinitis cases are caused by allergic reactions.Rhinitis is also produced as a response to chemical exposures includingcigarette smoke, temperature changes, infections and other factors.

Another study knocked down ORAI1 with siRNA to attenuatehistamine-mediated COX-2 expression and NFkB activation, indicating thatORAI-mediated NFkB activation was an important signaling pathwayresponsible for transmitting histamine signals that trigger inflammatoryreactions in allergic responses {Huang WC et al 2011}.

Therefore, it is likely that an important part of the inflammatoryresponse in ocular allergy is mediated by ORAI1 activation.

There are patent documents referring to siRNA directed to knockdown ofORAI1 for the treatment of an allergy. WO2010/099401 (The Board ofTrustees of the Leland Stanford Junior University) describes a method ofmodulating activity of a CRAC channel in a cell wherein a CRAC channelis contacted with an amino acid residue STIM1 domain that binds to ORAI1and opens the CRAC channel. Among the treatable conditions are listed:allergy, or hypersensitivity, of the immune system, including delayedtype hypersensitivity and asthma. In the description it is indicatedthat siRNA might be used to disrupt the expression of an endogenous geneto determine whether the endogenous gene has an effect on modulating theinteraction between STIM and ORAI proteins.

WO2007/121186 (The Queen's Medical Center) describes siRNA-mediatedsilencing of human CRACM1 (ORAI1) in human embryonic kidney cells(HEK293). It also indicates that agents that modulate CRAC channelactivity via interaction with CRACM1 (ORAI1) protein or disruption ofCRACM1 (ORAI1) expression can be used to modulate allergic reactions.

WO2009/095719 (ISIS INNOVATION LIMITED) describes methods, uses andproducts for use in treating disorders associated with mast cellactivation, including combinations of agents which inhibit theCRACM1(ORAI1) protein, which may be siRNAs, and agents which inhibitleukotriene activation of mast cells for use in the treatment of anallergic disorder, specifically allergic rhinitis.

US2011/0112058 (Incozen Therapeutics Pvt. Ltd.) describes a method foridentifying a candidate agent for treating lung cancer which preferablyinhibits CRACM1/ORAI1. This document describes also the use of siRNA tomodulate the CRAC/STIM pathway. It also indicates that CRAC channelmodulators have been said to be useful in treatment, prevention and/oramelioration of diseases or disorders associated with calciumrelease-activated calcium channel, including allergic rhinitis andallergic conjunctivitis.

US2012/0264231 (Hogan Patrick et al) describes methods and systems foridentifying an agent, which may be siRNA, which modulates calcium fluxthrough the ORAI channel and/or regulates intracellular calcium via theORAI channel, for the treatment of conditions and diseases associatedwith disregulation of calcium signaling, including allergic rhinitis orallergic conjunctivitis.

an important part of the inflammatory response in ocular allergy ismediated by

ORAI1 not only is a key determinant of the inflammatory response, but itis also related to cell proliferation and cell migration. The inhibitionof ORAI1 by a siRNA in retinal pigment epithelia (RPE) cells showed thatORAI1 is involved in epidermal growth factor (EGF)-mediated cell Thisgrowth. study hypothesized that ORAI1 might be a potential therapeutictarget for drugs aimed at treating EGF-related disorders, such asproliferative vitreoretinopathy (PVR) {Yang et al 2013}. PVR is adisease that develops as a complication of rhegmatogenous retinaldetachment, during which fluid from the vitreous humor enters a retinalhole. The mechanisms by which retinal holes or tears are formed are notfully understood yet. The accumulation of fluid in the subretinal spaceand the tractional force of the vitreous on the retina result inrhegmatogenous retinal detachment. During this process the retinal celllayers come in contact with vitreous cytokines that trigger the abilityof the RPE to proliferate and migrate undergoing epithelial-mesenchymaltransition (EMT) and develop the ability to migrate out into thevitreous. During this process the RPE cell layer-neural retinal adhesionand RPE-ECM (extracellular matrix) adhesions are lost. The RPE cells laydown fibrotic membranes while they migrate and these membranes contractand pull at the retina. All these finally lead to secondary retinaldetachment after primary retinal detachment surgery.

SUMMARY OF THE INVENTION

The present invention provides improved products for reducing ORAI1expression and consequent ocular inflammation in ocular allergies. Theadvantage of treating ocular allergies with siRNA products versustraditional anti-allergic therapeutic agents and allergyimmunotherapeutic drugs is that treatments based on siRNA will have alonger-lasting effect. This result is due to the fact that once theeffector molecule is no longer present, the cell will have to synthesisenew receptors from scratch; whereas traditional treatments would leavethe levels of receptors on the cell membrane intact.

Ocular allergies appear to be on the rise worldwide. Particularly inindustrialized nations, environmental pollution is widely considered amajor contributor to the heightened sensitivity of allergic individuals.In addition to worsening emissions pollution, studies have also pointedto a global increase in airborne allergens. Still another considerationis that residents of poorer countries are less likely to seek treatmentfor ocular allergies, a factor which may keep the reported incidence ofthe disease artificially low in underdeveloped countries.

Asthma and Allergy Foundation in America (AAFA) indicated that the USannual cost of allergies is estimated to be nearly $14.5 billion. Theyestimated 50 million Americans suffer from all types of allergies (1 in5 Americans) including indoor/outdoor, food & drug, latex, insect, skinand eye allergies. US allergy prevalence overall has been increasingsince the early 1980s across all age, sex and racial groups.

Despite geographic peculiarities, physicians from around the world findcommon ground in their criteria for choosing an appropriate treatmentcourse. These criteria include efficacy, safety, and convenience ofdosing and comfort of administration for the patient, according tospecialists from several countries. Therefore, with an increasing numberof patients complaining of a range of ocular allergic symptomsworldwide, finding the optimal treatment is every day both more complexand more interesting.

DESCRIPTION OF THE DRAWINGS

FIG. 1: shows short fragments of the target gene sequence ORAIl chosenas the target sequences of the siRNAs of the present invention

FIG. 2: shows oligonucleotide sequences for siRNA molecules of thepresent invention targeting ORAI1 encompassed by the present invention.The SEQ ID NOs given in the Figure refer to the sense (5′->3′) strand;typically siRNAs will be administered as dsRNAs, so will include boththe sense strand and its complement antisense strand. SEQ ID NO. 112 toSEQ ID NO. 222 are siRNAs targeting SEQ ID NO. 1 to SEQ ID NO. 111,respectively. Generally, an siRNA will include the sense and antisensestrand, and may also include 3′ dinucleotide overhangs (for example,dTdT). However, this is not essential.

FIG. 3: modified siRNAs targeting ORAIl. The SEQ ID NOs given refer tothe sense (5′->3′) strand of the modified ORAIl siRNAs.

FIG. 4: in vitro ORAIl expression levels after transfection of SEQ IDNO. 112 in different cell lines (human A204, murine C2Cl2 and murineJ744A,1).

FIG. 5: in vitro ORAIl expression levels after transfection of siRNAstargeting ORAI1 in murine C2Cl2 cell line.

FIG. 6: in vitro toxicity levels of different cell lines aftertransfection of SEQ ID NO. 112 in different cell lines (human A204 andmurine C2Cl2).

FIG. 7: in vitro human ORAIl expression levels after transfection of SEQID NO. 112 and its modified counterparts, SEQ ID NO. 223, SEQ ID NO.224, SEQ ID NO. 225, SEQ ID NO. 226, SEQ ID NO. 227, SEQ ID NO. 228, andSEQ ID NO. 229, in human A204 cells.

FIG. 8: in vitro murine ORAI1 expression levels after transfection ofSEQ ID NO. 112 and its modified counterparts, SEQ ID NO. 223, SEQ ID NO.224, SEQ ID NO. 225, SEQ ID NO. 226, SEQ ID NO. 227, SEQ ID NO. 228, andSEQ ID NO. 229, in murine C2C12 cells.

FIG. 9: shows the dose response of SEQ ID NO. 112 (SYL116011) and SEQ IDNO. 227 (SYL116011v8) in human cells. Transfections of Human A204 cellswith increasing doses (0.001 to 100 Nm) of SEQ ID NO. 112 (SYL116011)and SEQ ID NO. 227 (SYL116011v8) and quantification of % ORAI1 geneexpression consequence of siRNA mechanism of action.

FIG. 10: shows the dose response of SEQ ID NO. 112 (SYL116011) and SEQID NO. 227 (SYL116011v8) in murine cells. Transfections of murine C2C12cells with increasing doses (0.001 to 100 Nm) of SEQ ID NO. 112(SYL116011) and SEQ ID NO. 227 (SYL116011v8) and quantification of %ORAI1 gene expression consequence of siRNA mechanism of action.

FIG. 11: shows the expression of ORAI1 and its paralogues ORAI2 andORAI3 after transfection of SEQ ID NO. 112 (SYL116011) and SEQ ID NO.227 (SYL116011v8) in human cells. Transfection of SEQ ID NO. 112 and SEQID NO. 227 in human A204 cells and quantification of % ORAI1, ORAI2 andORAI3 gene expression consequence of siRNA mechanism of action.

FIG. 12: shows the expression of ORAI1 and its paralogues ORAI2 andORAI3 after transfection of SEQ ID NO. 112 (SYL116011) and SEQ ID NO.227 (SYL116011v8) in murine cells. Transfection of SEQ ID NO. 112 andSEQ ID NO. 227 in C2C12 murine cells and quantification of % ORAI1,ORAI2 and ORAI3 gene expression consequence of siRNA mechanism ofaction.

FIG. 13: shows the expression of putative OTEs after transfection of SEQID NO. 112 (SYL116011) in human cells. Transfection of SEQ ID NO. 112 inhuman A204 cells and quantification of % ORAI1, MSLN and OLFM12A geneexpression consequence of siRNA mechanism of action.

FIG. 14: shows ORAIl expression levels after transfection of SEQ ID NO.112 (SYL116011) in rat cell lines. Transfection of SEQ ID NO. 112 in ratC6 cells and quantification of % ORAIl gene expression consequence ofsiRNA mechanism of action.

FIG. 15: shows ORAIl expression levels after transfection of SEQ ID NO.112 (SYL116011) in rat cell lines. Transfection of SEQ ID NO. 112 in ratJTC-19 cells and quantification of % ORAIl gene expression consequenceof siRNA mechanism of action.

FIG. 16: shows gene expression levels of ORAI1 after transfection of SEQID NO. 112 (SYL116011), SEQ ID NO. 233 (SYL116011v11) and SEQ ID NO. 235(SYL116011v11) in human, murine and rat cells.

FIG. 17: Schedule of the in vivo assay.

FIG. 18: Levels of ORAIl mRNA in mouse whole eye at different timesfollowing induction of ocular allergy. NA: no allergy.

FIG. 19: mRNA levels of TLSP and Tnfrsf9 in a mouse model ofragweed-pollen induced allergy. mRNA levels are expressed as percentageof the levels observed prior to induction of allergy.

FIG. 20: Ocular clinical signs indicative of ocular allergy. Mice wereobserved 1, 3, 6 and 24h after induction of ocular allergy. Clinicalsigns were assessed by grading the following parameters on a scale 0-3:conjunctival chemosis and injection, hyperemia, lid edema, discharge andtearing. Data are expressed as percentage of the clinical scoring at 1 hafter induction of allergy of the PBS treated group and represent means±s.e.m of animals for PBS and 15 animals for the SEQ ID NO. 112(SYL116011) treated groups.

FIG. 21: Chemosis and tearing in response to treatment with differentdoses of SEQ ID NO. 112 (SYL116011) in a mouse model of ragweed-polleninduced allergy. Mice were observed 1, 3, 6 and 24 h after induction ofocular allergy. A) Conjunctival chemosis and B) tearing were scored on ascale 0-3. Data are expressed as percentage of scoring at 1 h afterinduction of allergy of the PBS treated group and represent means of 8animals for PBS and 15 animals for the SEQ ID NO. 112 (SYL116011)treated groups.

FIG. 22: Infiltration of mast cells in palpebral and bulbar conjunctivain response to treatment with different doses of SEQ ID NO. 112(SYL116011) in a mouse model of ragweed-pollen induced allergy. A)Infiltration of mast cells in palpebral conjunctiva expressed aspercentage of number of mast cells observed in PBS treated samples 3hafter treatment. B) Infiltration of mast cells in bulbar conjunctivaexpressed as percentage of number of mast cells observed in PBS treatedsamples 3h after treatment.

FIG. 23: Infiltration of eosinophils in palpebral and bulbar conjunctivain response to treatment with different doses of SEQ ID NO. 112(SYL116011) in a mouse model of ragweed-pollen induced allergy. A)Infiltration of eosinophils in palpebral conjunctiva expressed aspercentage of number of mast cells observed in PBS treated samples 24hafter treatment. B) Eosinophil infiltration in bulbar conjunctivaexpressed as percentage of number of eosinophils observed in PBS treatedsamples 24h after treatment.

FIG. 24: TLSP and CD-137 expression in response to treatment withdifferent doses of SEQ ID NO. 112 (SYL116011) in a mouse model ofragweed pollen induced-allergy. A) Expression of TLSP; B) Expression ofCD-137 (Tnfrsf9).

FIG. 25: Clinical signs observed at different time-points after allergychallenge. Allergy was induced by administering an ocular dose ofragweed pollen to mice pretreated with PBS, SEQ ID NO. 227 (SYL116011v8)or levocabastine. Data represent means of 10 animals per group.

FIG. 26: Change from Post-Dose Hyperemia—SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

FIG. 27: Change from Post-Dose Squinting—SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

FIG. 28: Change from Post-Dose Lid Swelling—SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

FIG. 29: Change from Post-Dose Discharge—SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to the provision of asiRNA molecule for use as a medicament, in the treatment and/orprevention of an eye condition characterised by increased expressionand/or activity of ORAI1, wherein said molecule specifically targets asequence selected from the group consisting of: SEQ ID NO. 1-SEQ ID NO.111 and reduces expression of the ORAI1 gene when introduced in a cell.Preferably the target sequence is selected from the group consisting ofSEQ ID NO. 1-SEQ ID NO. 14, more preferably the group consisting of SEQID NO. 1-SEQ ID NO. 8, and even more preferably the target sequencecomprises or consist of SEQ ID NO. 1.

A gene is “targeted” by a siRNA according to the present invention when,for example, the siRNA molecule selectively decreases or inhibits theexpression of the gene. The phrase “selectively decrease or inhibit” asused herein encompasses siRNAs that affect expression of one gene, inthis case ORAI1. Alternatively, a siRNA targets a gene when (one strandof) the siRNA hybridizes under stringent conditions to the genetranscript, i.e. its mRNA. Hybridizing “under stringent conditions”means annealing to the target mRNA region under standard conditions,e.g., high temperature and/or low salt content which tend to disfavourhybridization. A suitable protocol (involving 0.1×SSC, 68° C. for 2hours) is described in Maniatis, T., et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 1982, at pages387-389.

Nucleic acid sequences cited herein are written in a 5′ to 3′ directionunless indicated otherwise. The term “nucleic acid” refers to either DNAor RNA or a modified form thereof comprising the purine or pyrimidinebases present in DNA (adenine “A”, cytosine “C”, guanine “G”, thymine“T”) or in RNA (adenine “A”, cytosine “C”, guanine “G”, uracil “U”).Interfering RNAs provided herein may comprise “T” bases, for example at3′ ends, even though “T” bases do not naturally occur in RNA. In somecases these bases may appear as “dT” to differentiatedeoxyribonucleotides present in a chain of ribonucleotides.

The target sequence as defined above is described as a target DNAsequence as used for definition of transcript variants in databases usedfor the purposes of designing siRNAs, whereas the specific compounds tobe used will be RNA sequences defined as such.

An expert in the field can access any target gene sequence throughpublic data bases. For example, the GenBank Accession Numbercorresponding to human ORAI1 mRNA is NM_032790 (Gene ID: 84876).Homologous GenBank Accession Number corresponding to mouse ORAI1 mRNA isNM_175423 (Gene ID: 109305). Furthermore, ENSEMBL (MBL-EBI/WellcomeTrust Sanger Institute) has the following ORAIl human and mouseAccession Number: ENSG00000182500 and ENSMUSG00000049686, respectively.The public transcripts for human ORAI1 mRNA are ENST00000330079 andENST00000537188.

Said preferred target region identified by the present inventioncomprises or consists of at least one sequence selected from the groupconsisting of SEQ ID NO. 1-SEQ ID NO. 111.

In a preferred embodiment, said preferred target region comprises orconsists of at least one sequence selected from the group consisting ofSEQ ID NO. 1-SEQ ID NO. 14.

In another preferred embodiment, said preferred target region comprisesor consists of at least one sequence selected from the group consistingof SEQ ID NO. 1-SEQ ID NO. 8. These sequences present 100% homologybetween the following species: Homo sapiens, Mus musculus, Canis lupusfamiliaris, and Rattus norvegicus.

In the RNAi field, when in vitro studies demonstrated that a human siRNAis not able to induce knock down of the animal model gene, a surrogatecompound (animal-active analogue) is synthetized in order to analyze theefficacy of the siRNA in the relevant animal model. This surrogate isdesigned against the same region as the human siRNA, thus the two siRNAshave the same sequence except for a few nucleotides, depending on thehomology between the human and the rabbit target gene. This approach hasbeen widely used for development of other oligonucleotides, specificallyfor toxicology studies {Kornbrust D. et al. 2013}.

In a more preferred embodiment, said preferred target region comprisesor consists of SEQ ID NO. 1 (5′-TGATGAGCCTCAACGAGCA-3′).

Consequently, a siRNA according to the aspects of the present inventionwill preferably comprise a double stranded RNA molecule, whose antisensestrand will comprise an RNA sequence substantially complementary to atleast one sequence selected from the group consisting of SEQ ID NO.1-SEQ ID NO. 111, and whose sense strand will comprise an RNA sequencecomplementary to the antisense strand, wherein both strands arehybridised by standard base pairing between nucleotides. Morepreferably, a siRNA according to aspects of the present invention willpreferably comprise a double stranded RNA molecule, whose antisensestrand will comprise an RNA sequence substantially complementary toselected from the group consisting of SEQ ID NO. 1-SEQ ID NO. 8, andeven more preferably consisting of SEQ ID NO. 1.

Within the meaning of the present invention “substantiallycomplementary” to a target mRNA sequence, may also be understood as“substantially identical” to said target sequence. “Identity” as isknown by one of ordinary skill in the art, is the degree of sequencerelatedness between nucleotide sequences as determined by matching theorder and identity of nucleotides between sequences. In one embodimentthe antisense strand of an siRNA having 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity to thetarget mRNA sequence are considered substantially complementary and maybe used in the present invention. The percentage of complementaritydescribes the percentage of contiguous nucleotides in a first nucleicacid molecule that can base pair in the Watson-Crick sense with a set ofcontiguous nucleotides in a second nucleic acid molecule. In a preferredembodiment, the antisense siRNA strand is 100% complementary to thetarget mRNA sequence, and the sense strand is 100% complementary to theantisense strand over the double stranded portion of the siRNA. ThesiRNA may also include unpaired overhangs, for example, 3′ dinucleotideoverhangs, preferably dTdT.

In a preferred embodiment, said eye condition identified by the presentinvention is an ocular allergy and/or ocular conjunctivitis. Morepreferably, said eye condition is selected from seasonal allergicconjunctivitis, perennial allergic conjunctivitis, vernalkeratoconjunctivitis, atopic keratoconjunctivitis, giant papillaryconjunctivitis, dry eye syndrome and combinations thereof.

As is known from the state of the art, many different structures havebeen proposed to achieve RNA interference. Generally these doublestranded molecules are from about 19 to about 25 nucleotides in length,and include blunt-ended structures as well as those with overhangs.Overhangs have been described to be advantageous and may be present onthe 5′ ends or on the 3′ ends of either strand as they reducerecognition by RNAses and imitate Dicer's natural substrate. Someauthors recommend including overhangs on both 3′ ends of the molecules,whereas others consider one overhang to be sufficient. Others havedescribed the use of blunt-ended structures with specific modificationpatterns (EP 1527176, WO 2005/062937, WO 2008/104978, EP 2322617, EP2348133, US 2013/0130377, and many others).

Overhangs may be comprised of between 1 and 5 nucleotides; typicallyoverhangs are made up of dinucleotides. Classical molecules used in thefield, comprise a 19 nucleotide double stranded molecule which furthercomprises 3′ dinucleotide overhangs preferably comprisingdeoxynucleotides as taught in initial studies by Tuschl (WO02/44321).These overhangs are said to further enhance resistance to nuclease(RNase) degradation. Later, Kim et al 2005 describe that 21-mer products(containing dinucleotide overhangs) are necessary for loading onto RISC.Further, Bramsen et al. 2009, describe the introduction of possibledestabilizing modifications to the overhangs to further increasesilencing efficiency.

As such, a preferred embodiment of the various aspects of the presentinvention refers to siRNA molecules targeting at least one sequenceselected from the group consisting of SEQ ID NO. 1-SEQ ID NO. 111 whichcomprise at least one overhang. More preferably, said siRNA moleculestarget at least one sequence selected from the group consisting of SEQID NO. 1-SEQ ID NO. 8, and even more preferably consisting of SEQ IDNO. 1. Where the invention relates to an siRNA molecule targeting atleast one sequence selected from SEQ ID NO. 1 to SEQ ID NO. 111, thesiRNA will include an antisense strand of equivalent length andcomplementary to the target, and a sense strand of equivalent length andcomplementary to the antisense strand. The antisense and sense strandsmay further include additional bases which are not complementary to theother strand or the target, and/or which are not paired in the doublestranded portion of the siRNA. For example, SEQ ID NO 1 is a 19,nucleotide sequence; the siRNA may include a 19, bp double strandedregion over this portion of sequence identity, and dinucleotideoverhangs.

A preferred embodiment of the various aspects of the present inventionrefers to siRNA molecules targeting at least one sequence selected fromthe group consisting of SEQ ID NO. 1-SEQ ID NO. 111, wherein each strandof the double-stranded siRNA molecules is about 18 to about 28 or more(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 or more)nucleotides long.

Another preferred embodiment of the various aspects of the presentinvention refers to siRNA molecules of 18-28 nucleotides long or moreand comprising a nucleotide sequence selected from the group consistingof SEQ ID NO. 112-SEQ ID NO. 229. More preferably, the double-strandedsiRNA molecules are at least 19 nucleotides long and selected from thegroup consisting of SEQ ID NO. 112-SEQ ID NO. 229.

Another alternative embodiment of the various aspects of the presentinvention provides blunt-ended molecules.

Further, a preferred embodiment of the present invention relates to ansiRNA comprising or consisting of a 19 nucleotide double-strandedstructure targeting at least one sequence selected from the groupconsisting of SEQ ID NO. 1-SEQ ID NO. 111. More preferably, the siRNAcomprising or consisting of a 19 nucleotide double-stranded structuretargeting at least one sequence selected from the group consisting ofSEQ ID NO. 1-SEQ ID NO. 8, and even more preferably consisting of SEQ IDNO. 1.

A particular embodiment of the present invention relates to a 19nucleotide double-stranded blunt-ended siRNA targeted against at leastone sequence selected from the group consisting of SEQ ID NO. 1-SEQ IDNO. 111. More preferably, the siRNA is targeted against at least onesequence selected from the group consisting of SEQ ID NO. 1-SEQ ID NO.8, and even more preferably consisting of SEQ ID NO. 1. In a furtherparticular embodiment this compound comprises or consists of at leastone sequence selected from the group consisting of SEQ ID NO: 112-SEQ IDNO. 229. In a further preferred embodiment, the antisense strand of thissiRNA is at least 80%, preferably at least 90%, complementary to atleast one sequence selected from the group consisting of SEQ ID NO.1-SEQ ID NO. 111.

In a preferred embodiment, this compound comprises or consists of atleast one sequence selected from the group consisting of SEQ ID NO.112-SEQ ID NO. 119.

In a more preferred embodiment, this compound comprises or consists ofSEQ ID NO. 112 (5′-UGAUGAGCCUCAACGAGCA-3′), corresponding to the sensestrand of our referenced compound named SYL1160011.

Furthermore, as described in the section termed background of the art,an important issue with siRNA molecules is their instability inbiological fluids due to the ubiquitous nature of RNAses. Consequently,the use of many different chemical modifications to nucleotides has beendescribed with the purpose of enhancing compound stability.

Another inherent problem of siRNA molecules is their immunogenicity,whereby siRNAs have been found to induce unspecific activation of theinnate immune system, including up-regulation of certain cytokines, e.g.type I and/or type II interferon as well as IL-12, IL-6 and/or TNF-alphaproduction. The origin of these effects is thought to be activation ofToll-like receptors such as TLR7, TLR8 and/or TLR3 by siRNA.

Both of these effects, recognition by RNases and immunogenicity, havealso been described to be sequence-dependent.

Some of the chemical modifications which enhance compound stability bydecreasing susceptibility to RNAses are also able to reduce induction ofimmune recognition of subsequent response. However, insertion ofchemically modified nucleotides in a siRNA may also result in decreasedsilencing efficacy as described in the previous section, and hence mustbe approached with caution.

Consequently, in a preferred embodiment of the various aspects of thepresent invention, the siRNA further comprises at least one nucleotidewith a chemical modification.

Preferred chemical modifications which enhance stability and reduceimmunogenic effects include 2′-O-methyl nucleotides, 2′-fluoronucleotides, 2′-amino nucleotides, 2′-deoxy nucleotides, or nucleotidescontaining 2′—O or 4′—C methylene bridges. Other preferred chemicalmodifications for exonuclease protection include ExoEndoLight (EEL):modification of all pyrimidines in the sense strand to 2′-O-methylresidues, and modifications of all pyrimidines in a 5′-UA-3′ or 5′-CA-3′motif in the antisense strand. In addition, position 1 of the sensestrand can also be changed to 2′-O-methyl, preventing 5′-phosphorylationof the sense strand and thus increasing specificity of the siRNA byfurther inactivating the sense strand. In addition, the sense strand canalso include a 2′-O-methyl modification in position 14, because 2′-O-Meat this position further inactivates the sense strand and thereforeincreases specificity of the siRNAs. In addition, other preferredchemical modifications for exonuclease protection include Methyl-Fluoro(MEF): exo-protection alternating 2′-fluoro and 2′-O-methylmodifications starting (5′-end) with a 2′—F on the sense strand andstarting with 2′-O-Me on the antisense strand. In addition, position 1of the sense strand can also be changed to 2′-O-Me and position 1 of theantisense strand to 2′—F (as this can efficiently be 5′-phosphorylated).Also, modification of the ribonucleotide backbone connecting adjacentnucleotides can be made by the introduction of phosphorothioate modifiednucleotides. A further preferred chemical modification within themeaning of the present invention relates to the substitution of uracylribonucleotides with deoxythymidine (deoxyribonucleotides). In anotherpreferred embodiment of the present invention, the at least onechemically modified nucleotide is on the sense strand, on the antisensestrand or on both strands of the siRNA.

Accordingly, in one embodiment, the siRNA comprises or consists of atleast one sequence selected from the group consisting of SEQ ID NO.223-SEQ ID NO. 229.

siRNA molecules as described above may be delivered to the cell interiorin their native structure using methods known in the art. For example,when studying in vitro gene silencing, these compounds are administeredusing standard transfection reagents. To achieve effects in vivo thesecompounds may also be administered naked or using delivery enhancingagents such as for example liposomes, conjugation with a specificmoiety, etc. although many different alternatives are known in the art,and are used differently depending on the desired target site within thebody.

Alternatively, siRNA molecules of the various aspects of the inventioncan be expressed within cells from eukaryotic promoters. Recombinantvectors capable of expressing the siRNA molecules can be delivered andpersist in target cells. Alternatively, vectors can be used that providefor transient expression of nucleic acid molecules. Such vectors can berepeatedly administered as necessary. Once expressed, the siRNA moleculeinteracts with the target mRNA and generates an RNA interferingresponse. The siRNA molecules produced in this manner are often termedshRNA (short hairpin RNA), as their sense and antisense strands arejoined by a small loop of nucleotides. Delivery of siRNA moleculeexpressing vectors can be systemic, such as by intravenous orintra-muscular administration, by administration to target cellsex-planted from a subject followed by reintroduction into the subject,or by any other means that would allow for introduction into the desiredtarget cell.

A further aspect of the invention relates to the use of siRNA targetingat least one sequence selected from the group consisting of SEQ ID NO.1-SEQ ID NO. 111 in the preparation of a medicament for use in a methodof treatment of an eye condition characterised by increased expressionand/or activity of ORAI1. More preferably, said at least one sequence isselected from the group consisting of SEQ ID NO. 1-SEQ ID NO. 8, andeven more preferably said at least one sequence consists of SEQ IDNO. 1. The method comprises inhibiting expression of ORAI1 in a patient.The term inhibition is used to indicate a decrease or downregulation ofexpression or activity. Preferably, the eye condition is an ocularallergy and/or conjunctivitis. In one embodiment, the eye condition isselected from the group comprising seasonal allergic conjunctivitis,perennial allergic conjunctivitis, vernal keratoconjunctivitis, atopickeratoconjunctivitis, giant papillary conjunctivitis, dry eye syndromeand combinations thereof.

Also provided is a method of treatment of an eye condition characterisedby increased expression and/or activity of ORAIl. The method comprisesinhibiting expression of ORAI1 in a patient. The method may compriseadministering siRNA targeting at least one sequence selected from thegroup consisting of SEQ ID NO. 1-SEQ ID NO. 111 More preferably, said atleast one sequence is selected from the group consisting of SEQ ID NO.1-SEQ ID NO. 8, and even more preferably said at least one sequenceconsists of SEQ ID NO. 1.

In some countries, the combination of chronic allergic conjunctivitisand dry eye syndrome is quite common. The increasing dry eye problem isdue to common artificial climatization, indoor and outdoor pollutantsand to other unknown reasons. Patients with dry eye syndrome are moreprone to suffer from ocular allergies since the tear film is animportant barrier in preventing allergens from coming into contact withmast cells.

Therapeutic treatment with siRNAs directed against ORAI1 mRNA isexpected to be beneficial over small molecule topical ocular drops byincreasing the length of time that effect is observed, thereby allowingless frequent dosing and greater patient compliance. This is especiallyimportant in cases such as ocular allergies and/or conjunctivitis,comprising but not limited to vernal keratoconjunctivitis, atopickeratoconjunctivitis, and giant papillary conjunctivitis, as they areoften chronic conditions.

Bearing in mind the preparation of such a medicament, the siRNA of thevarious aspects of the present invention may be formulated as apharmaceutical composition. Preferably, the compositions andformulations of said siRNAs may be administered topically to the organof interest. In an even more preferred embodiment they may be formulatedfor topical administration to the eye, preferably to the corneal surfaceof the eye. Application to the corneal surface may, for example be inthe form of eye drops, a gel, lotion, cream or ocular inserts. Otheradministration forms to the eye may include injection into the eye.

A further preferred embodiment of the various aspects of the presentinvention relates to an siRNA specifically targeting at least onesequence selected from the group consisting of SEQ ID NO. 1-SEQ ID NO.111 as described in the preceding paragraphs, for use as a medicamentfor the treatment of an eye condition characterised by increasedexpression and/or activity of ORAI1. More preferably, said at least onesequence is selected from the group consisting of SEQ ID NO. 1-SEQ IDNO. 8, and even more preferably said at least one sequence consists ofSEQ ID NO. 1. As described above, it may be an siRNA comprising orconsisting of a 19 nucleotide double-stranded structure targeting atleast one sequence selected from the group consisting of SEQ ID NO.1-SEQ ID NO. 111. This siRNA may be blunt-ended. Preferably, the siRNAcomprises or consists of at least one sequence selected from the groupconsisting of SEQ ID NO. 112-SEQ ID NO. 229. Other siRNA for useaccording to the invention comprises or consists of at least onesequence from the group consisting of SEQ ID NO. 223-SEQ ID NO. 229.

Within the context of the present invention, to “specifically target” asequence the siRNA of the invention preferably comprises at least thesame seed sequence. Thus, any sequence according to the invention thatspecifically targets at least one sequence selected from the groupconsisting of SEQ ID NO. 1-SEQ ID NO. 111 is preferably identical inpositions 2-8 of the antisense strand. More preferably, said at leastone sequence is selected from the group consisting of SEQ ID NO. 1-SEQID NO. 8, and even more preferably said at least one sequence consistsof SEQ ID NO. 1.

Notwithstanding the above, the siRNAs of the various aspects of thepresent invention may be used to silence ORAI1 expression in tissuesother than the eye. Consequently, said siRNAs should be formulatedaccordingly.

For example, a siRNA molecule can comprise a delivery vehicle, includingliposomes, for administration to a subject. Carriers and diluents andtheir salts can be present in pharmaceutically acceptable formulations.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins poly (lactic-co-glycolic) acid (PLGA) and PLCAmicrospheres, biodegradable nanocapsules, and bioadhesive microspheres,or by proteinaceous vectors. In one embodiment of the present invention,the siRNA molecule is delivered through a cell-specific siRNA carrierthat combines components of the hepatitis B virus and liposomes. Inanother embodiment, the nucleic acid molecules of the invention can alsobe formulated or complexed with polyethyleneimine and derivativesthereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine- polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. The preferred compositions of theinvention are aqueous solutions, specifically saline solutions such asphosphate-buffered saline (PBS) with a pH range of about 7.0 to about7.4, preferably with a pH of 7.2 ±0.5.

A siRNA molecule of the invention may be complexed with membranedisruptive agents and/or a cationic lipid or helper lipid molecule.

Delivery systems which may be used with the invention include, forexample, aqueous and non-aqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and non-aqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer.

A pharmaceutical formulation of the invention is in a form suitable foradministration, e.g., systemic or local administration, into a cell orsubject, including for example a human. Suitable forms, in part, dependupon the use or the route of entry, for example oral, transdermal, or byinjection. Other factors are known in the art, and includeconsiderations such as toxicity and forms that prevent the compositionor formulation from exerting its effect.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art. For example, preservatives, stabilizers, dyesand flavouring agents can be provided. These include sodium benzoate,sorbic acid and esters of p-hydroxybenzoic acid. In addition,antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) a disease state. The pharmaceuticallyeffective dose generally depends on the type of disease, the compositionused, the route of administration, the type of mammal being treated, thephysical characteristics of the specific mammal under consideration,concurrent medication, and other factors that those skilled in themedical arts will recognize.

A therapeutically effective amount may also refer to the amount of asiRNA sufficient to delay or minimize the onset of an eye disorderassociated with ocular allergy. A therapeutically effective amount mayalso refer to the amount of the therapeutic agent that provides atherapeutic benefit in the treatment or management of an eye disorderassociated with ocular allergy. Further, a therapeutically effectiveamount with respect to a siRNA of the invention means that amount oftherapeutic agent alone, or in combination with other therapies, thatprovides a therapeutic benefit in the treatment or management of an eyedisorder associated with ocular allergy. Used in connection with anamount of a siRNA of the invention, the term can encompass an amountthat improves overall therapy, reduces or avoids unwanted effects, orenhances the therapeutic efficacy of or synergizes with anothertherapeutic agent.

A therapeutic benefit in the treatment or management of an eye disordersuch as ocular allergy is the sustained decrease in allergic symptoms.Given that siRNA will decrease the levels of ORAI1 within the cell, oncethe treatment stops the cell must re-synthesise new proteins. As suchtherapies based on siRNA treatments will have a more sustained effect.This is considered a significant enhancement of the therapeuticefficacy.

An additional benefit of using siRNA is the minimum probability of sideeffects or acute toxicity issues derived from its presence in systemiccirculation, often associated with different eyedrop-based treatments.This is due to the fact that when the compound enters the bloodstream,it will be rapidly degraded by RNAses present in the blood.

On the other hand, the fact that the siRNA molecule can be marketed insingle dose vials means addition of antimicrobial preservatives to theformulation can be avoided. Preservatives are present in the majority offormulations on the market today. These preservatives can produceintolerance in some patients, making it necessary to stop the treatment.Both issues are especially important when bearing in mind thatconditions such as ocular allergies and/or conjunctivitis, comprisingbut not limited to vernal keratoconjunctivitis, atopickeratoconjunctivitis, and giant papillary conjunctivitis, are oftenchronic and therefore so is the treatment.

One of the preferred administration routes is topical, by instillationdirectly to the eye, preferably using eyedrops. As described above,therapeutic treatment with siRNAs directed against ORAI1 mRNA isexpected to be beneficial over small molecule topical ocular drops byincreasing the length of time that the effect is observed, therebyallowing less frequent dosing and greater patient compliance.

However, as explained above, administration routes other than directlyto the eye can also be used. The precise dosage and administrationschedule to be employed in the formulation will also depend on the routeof administration. A skilled person would understand that the precisedosage and administration schedule to be employed also depends on theseriousness of the disorder, and should be decided according to thejudgment of the practitioner and each patient's circumstances. It isalso understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

The formulations or siRNA of the invention and described herein can beadministered in unit dosage formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. Formulations can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs. Compositions intended for oral use can be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions can contain one ormore such sweetening agents, flavouring agents, colouring agents orpreservative agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients that aresuitable for the manufacture of tablets.

These excipients can be, for example, inert diluents; such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia; and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets can be uncoated or they can be coated by knowntechniques. In some cases such coatings can be prepared by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more colouringagents, one or more flavouring agents, and one or more sweeteningagents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavouring agents canbe added to provide palatable oral preparations. These compositions canbe preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavouring and colouringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavouring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavouringand colouring agent. The pharmaceutical compositions or siRNA of theinvention and described herein can be in the form of a sterileinjectable aqueous or oleaginous suspension.

This suspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above.

A sterile injectable preparation can also be a sterile injectablesolution or suspension in a non-toxic parentally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil can be employedincluding synthetic mono-or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In preferred embodiments, the compositions of the invention areformulated in a solution, preferably a buffered saline solution such asPBS, or a gel for topical administration to the eye, such as, forexample, in the form of eyedrops. In such embodiments, the formulationsmay be cationic emulsions and/or contain biopolymers including, but notlimited to, poly(lactide-co-glycolide), carbopol, hyaluronic acid andpolyacrylic acid.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e. g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

As such, a further preferred embodiment of the present invention relatesto a pharmaceutical composition wherein said composition comprises atleast an siRNA targeting at least one sequence selected from the groupconsisting of SEQ ID NO. 1-SEQ ID NO. 111, as has been described in thepreceding paragraphs. More preferably, said at least one sequence isselected from the group consisting of SEQ ID NO. 1-SEQ ID NO. 8, andeven more preferably said at least one sequence consists of SEQ ID NO.1.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

As used herein the terms “ocular allergy” refers to an allergic disorderof the ocular surface caused by increased expression and/or activity ofORAIl. It may also be called allergic conjunctivitis. Ocular allergyincludes a wide variety of pathological conditions including but notlimited to: seasonal allergic conjunctivitis (SAC), perennial allergicconjunctivitis (PAC), vernal keratoconjunctivitis (VKC), atopickeratoconjunctivitis (AKC), and giant papillary conjunctivitis (GPC).

As used herein the terms “conjunctivitis” refers to an inflammation ofthe conjunctiva. It is also called pink eye or madras eye in India. Itis commonly due to an infection (usually viral, but sometimes bacterial)or an allergic reaction.

“Clinical symptoms” of ocular allergy include but are not limited toocular itching, ocular redness, swelling of the eyelids, chemosis,tearing, and nasal inflammation, nasal congestion, rhinorrhea, nasalpruritis and ear/palate pruritis, and sneezing. It is preferred that thepresent invention treats or prevents at least two clinical symptoms,more preferably at least three, even more preferably more than four.

The term “patient,” as used herein, refers to animals, includingmammals, preferably humans.

As used herein the term “allergen” refers to any antigenic substance inthe environment that is capable of producing immediate hypersensitivity(allergy). The list of known allergens includes plant pollens, spores ofmold, animal dander, house dust, foods, feathers, dyes, soaps,detergents, cosmetics, plastics, and drugs. Allergens can enter the bodyby being inhaled, swallowed, touched, or injected. Airborne allergensare allergens that are light enough to be carried through air currents,for example but not limited to, pollen or spores.

The term “allergic conjunctivitis” in the present invention isunderstood as inflammation of the conjunctiva caused by an allergicreaction. The conjunctiva is a thin membrane that covers the eye. Whenan allergen irritates the conjunctiva, common symptoms that occur in theeye include: redness (mainly due to vasodilation of the peripheral smallblood vessels), ocular itching, eyelid swelling, increased lacrimation,photophobia, watery discharge, and foreign body sensation (with pain).Symptoms are usually worse for patients when the weather is warm anddry, whereas cooler temperatures and rain tend to assuage symptoms.

The term “blepharitis” in the present invention is understood as achronic inflammation of the eyelid.

The term “blepharoconjunctivitis” in the present invention is understoodas the simultaneous occurrence of two separate eye conditions:blepharitis and conjunctivitis. Blepharitis affects the outer eyelids,while conjunctivitis occurs in the conjunctiva.

The term “keratoconjunctivitis” in the present invention is understoodas the inflammation of the cornea and conjunctiva.

The invention is further described in the following non-limitingexamples.

EXAMPLES 0. Materials

-   -   Mouse ORAI1 Probe: Taqman Gene Expression Assay Mm00774349_m1.    -   Mouse TLSP Probe: Taqman Gene Expression Assay Mm01157588_m1.    -   Mouse TNFSR9 probe: Taqman Gene Expression Assay Mm00441899_m1.    -   18S Endogenous control: Taqman Gene Expression Assay.        Hs99999901_s1.    -   Multiscribe Reverse Transcriptase 50U/ml (Applied Biosystems P/N        4311235).    -   RNAse inhibitor 20U/μl (Applied Biosystems P/N N8080119).    -   TaqMan 2X Universal Master Mix.    -   Non Radioactive Cell Proliferation Assay kit (Promega, Mannheim,        Germany).    -   Human mast cells (HMC-1).    -   Ionomycin calcium salt 1mM in DMSO (from Sigma Life Science        Ref#I3909-1ml).    -   Annexin-V detection kit Life Technologies (Ref: V13241).        1. In vitro analysis        1.1 ORAI1 expression levels after transfection of siRNAs of the        present invention in different cell lines.

In order to demonstrate the silencing effect of the siRNAs of thepresent invention, in vitro ORAI1 expression levels were measured aftertransfection of a selection of siRNAs of the present invention indifferent cell lines. Human A204 and murine C2C12 and J744A.1 cells weretransfected with 100 nM of SEQ ID NO. 112, 19 bp blunt ended dsRNAstructure, with Transit TKO and Lipofectamine 2000 respectively astransfection agent. All transfections were performed following standardmanufacturer's instructions. In the same transfection a scrambled siRNAsequence was used as a control of the specificity of interference. Cellpellets were collected at 24, 48, and 72 hours after transfectionexperiment and processed to evaluate possible variations in mRNA levelsas a consequence of siRNA mechanism of action. RNA levels werequantified by real-time PCR using a relative quantitation method, theComparative Threshold 2-ΔΔ CT method. (Livak and Schmittgen, 2001). Allreal time quantitative PCR experiments were performed in triplicate andrepeated in three independent experiments. Mean and standard deviationwere calculated. As FIG. 4 shows, SEQ ID NO. 112 reduced significantlyORAI1 mRNA levels approximately 70-80% in A204 and C2C12 cells and 40%in J744A.1. SEQ ID NO. 113, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO.117, SEQ ID NO. 118 and SEQ ID NO. 119, 19 bp blunt ended dsRNAstructures, also significantly reduced ORAI1 mRNA expression levelsapproximately 40-80% (FIG. 5).

1.2 Cellular viability of different cell lines after transfection with asiRNA of the present invention.

In order to demonstrate the cellular viability of the siRNAs of thepresent invention, in vitro toxicity levels were measured aftertransfection of a selection of siRNAs of the present invention indifferent cell lines. Human A204 and murine C2C12 and J744A.1 cells weretransfected with 100 nM of SEQ ID NO. 112 (19 bp blunt ended dsRNAstructure) with Transit TKO and Lipofectamine 2000 respectively astransfection agent. All transfections were performed following standardmanufacturer's instructions. In the same transfection a scrambled siRNAsequence was used as a control of the specificity of interference. Cellpellets were collected at 24, 48, and 72 hours after transfectionexperiment and processed to evaluate possible variations in cellviability levels as a consequence of siRNA transfection. Cell viabilitywas measured using CellTiter 96® Aqueous Non-Radiactive Cell.Proliferation Assay from Promega. This method is based on capacity ofliving cells (dehydrogenase enzymes) to reduce the MTS tetrazoliumcompound into formazan product as measured by the amount of 490 nmabsorbance. Mean and standard deviation were calculated. As FIG. 6 showsno changes in cell viability levels were found for SEQ ID NO. 112.Therefore, SEQ ID NO. 112 is not toxic and it is safe.

1.3 ORAI1 expression levels after transfection of unmodified andchemically modified siRNA of the present invention in different celllines.

In order to improve the stability of siRNAs of the present invention andto ensure no immunogenic activation, different siRNA-optimized chemicalmodifications were introduced to the canonical SEQ ID NO. 112 sequence(19 bp blunt ended dsRNA structure); thus new chemically modifiedentities (SEQ ID NO. 223, SEQ ID NO. 224, SEQ ID NO. 225, SEQ ID NO.226, SEQ ID NO. 227, SEQ ID NO. 228 and SEQ ID NO. 229) were obtainedand transfected in human and murine cells to prove their ability toreduce ORAI1 mRNA levels. Chemical modifications are detailed in FIG. 3.Human A204 and murine C2C12 and J744A.1 cells were transfected with 100nM of SEQ ID NO. 112, SEQ ID NO. 223, SEQ ID NO. 224, SEQ ID NO. 225,SEQ ID NO. 226, SEQ ID NO. 227, SEQ ID NO. 228 or SEQ ID NO. 229 (allthese structures correspond to 19 bp blunt ended dsRNA structures) withTransit TKO and Lipofectamine 2000 respectively as transfection agent.All transfections were performed following standard manufacturer'sinstructions. In the same transfection a scrambled siRNA sequence wasused as a control of the specificity of interference. Cell pellets werecollected at 24, 48, and 72 hours after transfection experiment andprocessed to evaluate possible variations in mRNA levels as aconsequence of the siRNA-treatment. RNA levels were quantified byreal-time PCR using a relative quantitation method, the ComparativeThreshold 2ΔΔ CT method {Livak and Schmittgen, 2001}. All real timequantitative PCR experiments were performed in triplicate and repeatedin three independent experiments. Mean and standard deviation werecalculated. As FIG. 7 and FIG. 8 show, modified siRNAs showed excellentefficacy, comparable to SEQ ID NO. 112, both in human and murine celllines. Thus, chemically modified products SEQ ID NO. 223, SEQ ID NO.224, SEQ ID NO. 225, SEQ ID NO. 226, SEQ ID NO. 227, SEQ ID NO. 228 andSEQ ID NO. 229 reduced ORAI1 mRNA levels between 50-80%.

1.4 Dose response of SEQ ID NO. 112 and SEQ ID NO. 227 in human andmurine cells.

Human A204 and murine C2C12 cells were transfected with increasing dosesof SEQ ID NO. 112 (19bp blunt ended dsRNA structure, SYL116011) and SEQID NO. 227 (19 bp blunt ended dsRNA structure, SYL116011v8) (0.001 to100 nM) with Transit TKO and Lipofectamine 2000 respectively astransfection agent. All transfections were done following standardmanufacturer's conditions. In the same transfection a scrambled siRNAsequence was used as specific control of interference. Cell pellets werecollected and processed to evaluate possible variations in mRNA levelsas a consequence of siRNA mechanism of action. RNA levels werequantified by real-time PCR using a relative quantitation method, theComparative Threshold 2-ΔΔ C_(T) method. {Livak and Schmittgen, 2001}.All real time quantitative PCR experiments were performed in triplicateand repeated in three independent experiments. Mean and SEM werecalculated. As FIG. 9 shows, a significant reduction in ORAI1 levels wasobserved in human cells at the dose 0.5 nM. The maximum effect was seenin response to the dose 100 nM both SEQ ID NO. 112 and SEQ ID NO. 227.Small differences were observed for SEQ ID NO. 112 and SEQ ID NO. 227between the concentrations 10 to 50 nM. SEQ ID NO. 227 reduced ORAI1mRNA levels 60-80% while SEQ ID NO. 112 reduced ORAI1 mRNA levels40-60%. No differences were observed between the concentrations of 0.05to 0.001 nM. Using these data, the inhibitory concentration 50 (IC50)value was calculated to be 1.98 nM for SEQ ID NO. 227 and 5.3 nM for SEQID NO. 112.

As FIG. 10 shows, a significant reduction in ORAI1 levels was alsoobserved in murine C2C12 cells at the dose 2.5 nM. The maximum effectwas seen in response to the dose 50 nM both SEQ ID NO. 112 and SEQ IDNO. 227. Small differences were observed for SEQ ID NO. 112 and SEQ IDNO. 227 between the concentrations 5 to 100 nM. Both SEQ ID NO. 112 andSEQ ID NO. 227 reduced ORAI1 mRNA levels 70-80% No differences wereobserved between the concentrations of 0.1 to 0.001 nM. Using thesedata, the inhibitory concentration 50 (IC50) value was calculated to be1.98 nM for SEQ ID NO. 227 and 1.25 nM for SEQ ID NO. 112.

1.5 Expression of ORAI1 and its paralogues ORAI2 and ORAI3 aftertransfection of SEQ ID NO. 112 and SEQ ID NO. 227.

In order to demonstrate the specific silencing effect of the siRNAs ofthe present invention, in vitro ORAI1, ORAI2 and ORAI3 expression levelswere measured after transfection of a selection of siRNAs of the presentinvention in different cell lines. We analyzed the effect of SEQ ID NO.112 and SEQ ID NO. 227 on receptors of the ORAI channels family toanalyze its effect on proteins that are structurally and functionallyrelated to ORAI1 channel. mRNA levels of ORAI1, ORAI2, and ORAI3 wereassessed in human A204 and C2C12 murine cells after treatment with SEQID NO. 112 and SEQ ID NO. 227. Human A204 and murine C2C12 cells weretransfected with 100 nM of SEQ ID NO 112 and SEQ ID NO 227 with TransitTKO and Lipofectamine 2000 respectively as transfection agents. Alltransfections were done following standard manufacturer's conditions. Inthe same transfection a scrambled siRNA sequence was used as specificcontrol of interference. Cell pellets were collected at 24, 48, and 72hours after transfection experiment and processed to evaluate possiblevariations in mRNA levels as a consequence of siRNA mechanism of action.FIG. 11 and FIG. 12 show that SEQ ID NO. 112 and SEQ ID NO. 227 wereable to selectively decrease the levels of ORAI1 mRNA, 70-80%approximately in human cells, without significantly affecting mRNAlevels of ORAI2 or ORAI3 (FIGS. 11), and 60-70% in murine cells (FIG.12).

1.6 Expression of putative OTEs after transfection of SEQ ID NO. 112(SYL116011) in human cells.

In order to demonstrate the specific silencing effect of the siRNAs ofthe present invention regarding unintended targets, putative in silicooff-targets effects (OTEs) for SEQ ID NO. 112 were determined in humancell lines. MSLN and OLFM12A expression levels were measured aftertransfection of a selection of siRNAs of the present invention in humancell lines. we analyzed the effect of SEQ ID NO. 112 on MSLN and OLFM12Agene expression. mRNA levels of MSLN and OLFM12A were assessed in humanA204 cells after treatment with 100 nM of SEQ ID NO. 112 with TransitTKO as transfection agents. All transfections were done followingstandard manufacturer's conditions and with positive and negativecontrols. In the same transfection a scrambled siRNA sequence was usedas specific control of interference. Cell pellets were collected at 24,48, and 72 hours after transfection experiment and processed to evaluatepossible variations in putative OTEs mRNA levels as a consequence of SEQID NO. 112 mechanism of action. FIG. 13 shows that SEQ ID NO. 112 didnot decrease the levels of putative OTEs.

1.7 ORAI1 expression levels after transfection of SEQ ID NO. 112 in ratcell lines

In order to demonstrate the silencing effect of the SEQ ID NO. 112, invitro ORAI1 expression levels were measured after transfection of aselection of siRNAs of the present invention in different cell lines.Rat JTC-19 and C6 cells were transfected with 100 nM of SEQ ID NO. 112with Transit IT2020 and Lipofectamine 2000 respectively, as transfectionagents. All transfections were done following standard manufacturer'sconditions with a negative control. In the same transfection a scrambledsiRNA sequence was used as specific control of interference. Cellpellets were collected at 24, 48, and 72 hours after transfectionexperiment and processed to evaluate possible variations in mRNA levelsas a consequence of siRNA mechanism of action. RNA levels werequantified by real-time PCR using a relative quantitation method, theComparative Threshold 2ΔΔ CT method {Livak and Schmittgen, 2001}. Allreal time quantitative PCR experiments were performed in triplicate andrepeated in three independent experiments. Mean and SEM were calculated.As FIG. 14 and FIG. 15 show SEQ ID NO. 122 reduced significantly ORAI1mRNA levels approximately 70% in JTC-19 cells and 40-70% in C6 cells.For SEQ ID NO. 112 ORAI1 mRNA levels are not completely recovered at 72hours in JTC-19 cells but not in C6 cells (FIG. 14 and FIG. 15).

1.8 Gene expression levels of ORAI1 after transfection of SEQ ID NO. 112(SYL116011), SEQ ID NO. 233 (SYL116011v11) and SEQ ID NO. 235(SYL116011v11).

Human A204, murine C2C12 and JTC-19 rat cells were transfected with 100nM of SEQ ID NO. 112 (19 bp blunt ended dsRNA structure, SYL116011) andSEQ ID NO. 233 (19 bp blunt ended dsRNA structure, SYL116011v11) and SEQID NO. 235 (19 bp blunt ended dsRNA structure, SYL116011v12 with TransitTKO, Lipofectamine 2000 AND Mirus IT2020, respectively as transfectionagent. All transfections were done following standard manufacturer'sconditions. In the same transfection a scrambled siRNA sequence was usedas specific control of interference. Cell pellets were collected andprocessed to evaluate possible variations in mRNA levels as aconsequence of siRNA mechanism of action. RNA levels were quantified byreal-time PCR using a relative quantitation method, the ComparativeThreshold 2ΔΔ C_(T) method. {Livak and Schmittgen, 2001}. As FIG. 16shows, substantial reductions in ORAI1 levels were observed in human,murine and rat cells. ORAI1 mRNA levels were reduced 70-80% in humancells for SEQ ID NO. 112 (SYL116011), SEQ ID NO. 233 (SYL116011v11) andSEQ ID NO. 235 (SYL116011v11). In murine cells, ORAI1 mRNA levels werereduced 50% for SEQ ID NO. 112 (SYL116011), SEQ ID NO. 233(SYL116011v11) and SEQ ID NO. 235 (SYL116011v11), while in rat cellswere reduced 50% for SEQ ID NO. 112 (SYL116011)and 95% and 99% for SEQID NO. 233 (SYL116011v11) and SEQ ID NO. 235 (SYL116011v11)respectively.

2. In vivo analysis2.1 Analysis of the efficacy in vivo of SEQ ID NO. 112 (SYL116011) andSEQ ID NO. 227 (SYL116011v8) in a mouse model of ocular allergy inducedby ragweed pollen.

The objective of the present study was to analyze the efficacy of thesiRNAs of the present invention designed to silence expression of ORAI1,specifically SEQ ID NO. 112 (19 bp blunt ended dsRNA structure,SYL116011) and SEQ ID NO. 227 (19 bp blunt ended dsRNA structure,SYL116011v8) to reduce symptoms associated with ocular allergies in amouse model of ocular allergy induced by ragweed pollen.

Ragweeds are flowering plants in the genus Ambrosia in the sunflowerfamily Asteraceae. Ragweed pollen is highly allergenic, generallyconsidered the greatest aeroallergen of all airborne pollens and theprime cause of hay fever worldwide. The National Institute ofEnvironmental Health Science (NIEHS) indicates that ragweed and otherweeds such as curly dock, lambs quarters, pigweed, plantain, sheepsorrel and sagebrush are some of the most prolific producers of pollenallergens around the world. This pollen is commonly used in animalmodels for studying allergic conjunctivitis {Bacsi A et al 2005}.

The aim of this analysis was to determine if down regulation of ORAI1 byocular instillation of compounds of the present invention (SEQ ID NO.112 (SYL116011) and SEQ ID NO. 227 (SYL116011v8)) alleviates thesymptoms caused by ragweed pollen-induced ocular allergy in mice.

We have analysed whether ORAI1 is expressed in the mouse eye and if itsexpression is up-regulated in response to ragweed pollen-induced ocularallergy. We have also assessed the effect of silencing the expression ofORAI1 using locally applied SEQ ID NO. 112 (SYL116011) or SEQ ID NO. 227(SYL116011v8) on allergy response in the above mentioned mouse model.For this purpose the following parameters have been analyzed:

-   -   Clinical signs in response to allergy induction: typical ocular        signs of allergic conjunctivitis include itching, eyelid        swelling, conjunctival swelling (chemosis), and mucus        deposition. Mucus associated to ocular allergies is profuse,        stringy and even sticky. Alterations to the conjunctiva usually        cause the bulbar conjunctiva to take on a “glassy” appearance        and the colouring of the palpebral conjunctiva is more pink than        red with a frequently milky appearance.    -   Number of local mast cells: minutes after allergic stimulation        conjunctival mast cells degranulate; the release of inflammatory        mediators attracts more mast cells that migrate from deeper        layers of the conjunctiva.    -   Local infiltration of eosinophils: infiltration of inflammatory        cells to the conjunctiva occurs hours after allergen exposure        and is part of the late response to allergens. Although several        different types of cells migrate to the conjunctiva the main        type are eosinophils.    -   Expression changes in molecular biomarkers related to allergy:        -   Thymic stromal lymphopoietin (TLSP) is an epithelium-derived            cytokine that activates dendritic cells by binding to its            specific receptor TLSPR. Binding of TLSP to TLSPR induces an            inflammatory Th2-type response. TLSP is produced primarily            by epithelial cells but can also be produced by mast cells            and has been found to be up-regulated at sites of allergic            inflammation {Zheng X. et al 2010}.        -   Tumor necrosis factor receptor superfamily, member 9            (Tnfrsf9) or CD-137 is a costimulator of memory T cells.            This costimulator is expressed in activated T cells, NK            cells and dendritic cells (DC), while its ligand CD137L has            been detected on mature DC, activated macrophages and            activated B cells. CD-137 costimulates T cell activation and            proliferation, enhances survival of activated T cells and            supresses CD4+ T help. In allergic inflammation it has been            shown to mediate IL-4 dependent Th2 responses and is            up-regulated in eosinophils of patients with IgE mediated            allergic responses.

A. Methods

a.1 Test system characterisation

TABLE 1 Test system characterisation Species: Mouse Strain: BALB-C Sex:Female Colour: White Rationale for selection This strain has beenpreviously been of species/strain: established as a good model forocular allergies {Bacsi A. et al 2005}. Approx. age of the animals 8-10weeks at the beginning of the study:

A further advantage of the siRNAs of the present invention is that SEQID NO. 1-SEQ ID NO. 8 correspond to highly conserved regions of theORAI1 gene, throughout different animal sequences. In fact, thesesequences are identical between human and mouse, making this animalmodel especially suitable for the study of for ocular allergies.

a.2 Induction of allergy

Allergic conjunctivitis was induced by immunizing the animals with amixture of 50 μg ragweed (Rw) pollen in 0.25 ml alum by intraperitonealinjection on day 1. The immunization solution was prepared immediatelyprior to administration and was protected from light at all times. Tendays after immunization 1.25 mg of Rw pollen was topically instilledinto each eye. Administrations were performed in a dose volume of 5μL/eye. This procedure was adapted from a standard preexisting publishedprotocol known to an expert in the field and validated prior toassessing the efficacy of the siRNAs {Magone M. T. et al 1998}.

a.3 Test item administration

The test item was applied by the topical ocular route to both eyes ofthe animals once a day over a period of 5 days starting on day 6 (FIG.4). A separate group of animals was administered with vehicle (PBS) andserved as control. Administrations were performed in a dose volume of 5μL/eye.

a.4 Clinical observations and collection of samples

General health status of animals was monitored daily from firstadministration until sacrifice. Mice were examined for clinical signs ofhypersensitivity prior to instillation of topical ocular pollen and atdifferent time-points up to 24 h after pollen instillation. Conjunctivalchemosis and injection, lid edema, discharge and tearing were graded ona scale 0-3. Clinical scoring was performed by an experimented observerblind to the experimental condition. Animals were sacrificed either 3 or24 h after allergy challenge. Prior to sacrifice a sample of blood wascollected in order to assess the presence of IgE, IL-13; IL-10 and MCP-1in plasma. Following sacrifice eyes, spleen and cervical lymph nodeswere isolated and either processed for histology, preserved in RNA lateror processed for analyzing the levels of the above mentioned cytokinesin conjunctiva.

a.5 Histopathology

The exenterated eyes were immersed in 10% formaline ( 1/20 volume) for24h hours, then the formaline was removed with several washes ofphosphate buffer 0.1 M and maintained almost 24 h hours in this buffer.Samples were dehydrated by incubating them in increasing concentrationsof ethanol, and were thereafter embedded in low melting paraffin in atissue processor (Leica TP 1020, Cat.no- 0704 37101, Leica Microsystems,Nussloch, Germany). Samples were cut in a microtome to obtain sectionsof 2 μm that were thereafter stained with either toludine blue to countthe number of mast cells or with hematoxyline-eosine to assesseosinophil infiltration.

a.6 RNA Isolation and retrotranscription

Total RNA was isolated from whole eyes, spleen or lymph nodes usingRNeasy RNA extraction kit (Invitrogen, Calif., USA). 4 μg of total RNAwere retrotranscribed using High-Capacity cDNA Archive kit (AppliedBiosystems, Inc., Foster City, Calif., USA) according to themanufacturer's instructions and the IT-B-0003-01.

a.7 qPCR

qPCR was performed using Stepone plus detection system (AppliedBiosystems). 500 nanograms of each sample were amplified in a TaqMan 2XUniversal Master Mix under the following conditions: 95° C. for 10 min,followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. All qPCRamplifications were performed in triplicate and repeated in at least twoindependent experiments, always including reverse transcription controlsand no template controls. ORAI1, TLSP and Tnfrsf9 mRNA levels wereanalyzed by qPCR using the ΔΔCT method of relative quantification using18S gene as internal standard {Livak K. J. and Schmittgen T. D., 2001}.

a.8 Analysis of IgE, IL-13; IL-10 and MCP-1 in plasma and conjunctiva

The amount of the following cytokines IgE, IL-13, IL-10 and MCP-1 wasassessed in plasma and conjunctiva of mice using the following kits andaccording to the manufacturer's instructions.

B. Results

b.1 Expression of ORAI1 in mouse eye and induction in response to ocularallergy.

Expression of ORAI1 was assessed in eyes of mice at different timepoints after induction of allergy as mentioned in the methods section.FIG. 17 shows that ORAI1 is present in the eye and that its expressionis rapidly up-regulated in response to the allergic challenge. Atwo-fold increase in ORAI1 mRNA levels was observed 3-6 h afteradministration of ragweed pollen. 24 h post challenge levels of ORAI1were approximately 350% of the basal levels.

b.2 Assessment of expression of allergy biomarkers in response to ocularallergy.

mRNA levels of TLSP and Tnfrsf9 were studied at different time-pointsfollowing induction of ocular allergy by instillation of ragweed pollenin pre-sensitized mice. A significant induction of both TLSP andTnfrsf9, was observed 3 h post challenge. 24 h after induction Tnfrsf9mRNA levels were close to baseline whereas mRNA levels of TLSP werestill approximately 1.5 times above basal levels (FIG. 18).

b.3 Efficacy of SEQ ID NO. 112 (SYL116011) in a mouse model of ocularallergy

Three groups of animals were intraperitoneally (IP) injected with a doseof ragweed pollen adsorbed on alum as mentioned in the methods section.Five days after the IP injection one group (A, n=8) received an ocularinstillation/day of PBS over a period of five days, the second groupreceived SEQ ID NO. 112 (SYL116011) at the dose of 150 μg/eye/day (lowdose) (B, n=15) during the same period of time whereas the third groupreceived SEQ ID NO. 112 (SYL116011) at the dose of 375 μg/eye/day (highdose) over 5 days. Animals were examined for symptoms related to ocularallergy 1, 3, 6 and 24 h after ocular instillation of pollen. As shownin FIG. 20, treatments with either dose of SEQ ID NO. 112 (SYL116011)significantly reduced the immediate clinical signs of allergy. Furtheranalysis of the clinical signs indicated that both doses of SEQ ID NO.112 (SYL116011) had a particular effect on two of the parametersstudied: chemosis (edema of the conjunctiva) and tearing (FIG. 21).

Infiltration of mast cells was assessed in palpebral and bulbarconjunctiva 3h after induction of ocular allergy. SEQ ID NO. 112(SYL116011) administered at the dose of 375μg/eye/day caused asignificant reduction in the number of mast cells infiltrating both thepalpebral and bulbar conjunctiva (FIG. 22).

Eosinophil infiltration was assessed in conjunctiva at 24h postchallenge. Again, a significant decrease in infiltrating eosinophils wasobserved in response to the high dose of SEQ ID NO. 112 (SYL116011) inboth regions of the conjunctiva and to the low dose in the bulbarconjunctiva (FIG. 23).

Analysis of the allergy biomarker TLSP in whole eye showed adose-dependent reduction of the expression of this marker in response toSEQ ID NO. 112 (SYL116011). Expression of CD-137 (Tnfrsf9) was alsosignificantly reduced in response to SEQ ID NO. 112 (SYL116011) 3 h postallergy challenge. As seen in FIG. 11 this allergy marker issignificantly induced 3h post allergy induction (FIG. 24).

b.4 Efficacy of SEQ ID NO. 227 (SYL116011v8) in a mouse model of ocularallergy

Furthermore, another in vivo experiment was performed in which threegroups of animals were intraperitoneally (IP) injected with a dose ofragweed pollen adsorbed on alum as mentioned in the methods section.Five days after the IP injection one group (A, n=10) received an ocularinstillation/day of PBS over a period of five days, the second groupreceived another compound of the present invention (SEQ ID NO. 227(SYL116011v8)), at the dose of 450μg/eye/day (B, n=10) during the sameperiod of time whereas the third group received 2 μL of 0.5 mg/mllevocabastine over 5 days. Levocabastine is a second generation H1receptor antagonist currently marketed for the treatment of ocularallergies. Animals were examined for symptoms related to ocular allergy0.5, 1, 3, 6 and 24 h after ocular instillation of pollen. As shown inFIG. 25, treatment with SEQ ID NO. 227 (SYL116011v8) significantlyreduced clinical signs of allergy; the reduction of clinical signs wasgreater to the one observed in response to levocabastine. Furtheranalysis of the clinical signs indicated that SEQ ID NO. 227(SYL116011v8) improves all the parameters studied when compared to PBStreated animals. Therefore this compound has proven to be an effectivetherapeutic treatment for ocular allergies.

2.2 Evaluation of the effects in vivo of SEQ ID NO. 112 (SYL116011) in amurine model for experimental allergic conjunctivitis.

The objective of the present study was to evaluate the effects of thesiRNAs of the present invention designed to silence expression of ORAI1,to reduce allergic symptoms like hyperemia, squinting, discharge, andlid swelling, associated to allergic conjunctivitis in a murine model.

The aim of this study was to evaluate if down regulation of ORAI1 byocular instillation of SEQ ID NO. 112 (19 bp blunt ended dsRNAstructure, SYL116011) alleviates allergic symptoms (hyperemia,squinting, discharge, and lid swelling) in a murine model of allergicconjunctivitis. As a positive control it was used a commonly used drug,the anti-allergic Patanol®. Patanol® (0.1% Olopatadine) is ananti-histamine/mast cell stabilizer dual-action administered as eyedrops. Patanol® blocks the effects of histamine and prevents mast cellsfrom releasing the chemicals responsible for allergy symptoms. Topicaladministration of PBS (vehicle) was used as a negative control.

A. Methods

In this study, female Balb/C mice were sensitized with short ragweedmixed with aluminum hydroxide on Day 0. On Day 18, mice were topicallysensitized with short ragweed in balanced salt solution (BSS) prior totopical treatment to eliminate mice that were considered non-respondersto challenge. Non-responders were mice that did not have at least a 2unit change from baseline in hyperemia. Due to a low number ofresponders, mice were sensitized again on Day 21. On Day 24, theprocedure for Day 18 was repeated in order to identify responders. Fortyeight mice were chosen for the study and randomized into 6 groups, with8 mice per group. Prophylactic treatment groups received theirrespective topical drugs on Days 25-27 (once daily for groups 2, 3 and5; four times daily for group 6). On Days 28-31, mice were topicallychallenged with ragweed twice daily while receiving their respectivedrug (once daily for all groups except the Patanol® group, whichreceived three doses daily). Animals were evaluated for hyperemia,squinting, discharge, and lid swelling with evaluations after the first,fourth, sixth, and eighth challenges.

a.1 Animals

The mice were housed in polycarbonate cages with direct contact bedding(ALPHA-dri®). The cages conformed to standards set forth in the AnimalWelfare Act and the Guide for the Care and Use of Laboratory Animals.Space recommendations for animals were in accordance with PHS policy andthe AWA. Litter or bedding in animal cages was changed as often asnecessary to keep animals dry and clean.

Animals were fed food that is fresh, palatable and nutritionallyadequate ad libitum. Water that is clean, potable, and uncontaminatedwas provided ad libitum. Environmental controls were set to maintaintemperatures 22±4° C. (68±5° F.) with relative humidity of 50±20%. A12-hour light/dark cycle was maintained. The animals were acclimated forat least 5 days after arrival at the facility prior to baselineevaluation. Staff veterinarian was not needed throughout the study.

a.2 Allergen Sensitization

-   -   Route: Subcutaneous, both hind hocks.    -   Frequency: On Day 0 and Day 21 for all groups.    -   Procedure: For each sensitization, animals receiving SRW        received 100 μg ragweed in 0.65 mg of aluminum hydroxide in 50        μL.

a.3 Dosing

Topical treatment with SEQ ID NO. 112 (SYL116011), Patanol® as positivecontrol or vehicle control was administered to all groups as outlined.Mice were dosed topically to the cornea using a calibrated micropipette,with a 3 μL drop of treatment in each eye. During the prophylactictreatment days (Days 25-27 Patanol® animals were dosed four times dailyat approximately 9 am, 12 pm, 2 pm and 5 pm. All times were ±60 minutesand the exact timing of the dosing was noted in the study binder.Animals in groups 2, 3, 5 and 6 received their topical dose atapproximately 1pm ±60 minutes. On challenge days (Days 28-31), Patanol®mice were given three times daily dosing on challenge days atapproximately 9 am, 1pm, and 4 pm; however, the time of dose was ±90minutes. All other groups were dosed at approximately 1 pm. Again, theexact timing of dosing was recorded in the study binder.

a.4 Allergen Challenge

-   -   Route: Ocular application, both eyes.    -   Frequency: On Day 18, a screening SRW challenge was performed to        identify responders. Mice were evaluated at baseline, prior to        SRW challenge. Then 18±1 minutes post challenge, animals were        evaluated again. Due to a lack of responders, the study was        delayed. A second sensitization occurred on Day 21, and the        screening SRW challenge was repeated on Day 24. On Days 25-27        groups 2, 3, 5 and 6 began their respective prophylactic        treatments. On Days 28-31, the BID challenges occurred        approximately 30 minutes after the 1^(st) daily Patanol® dose        and after the 3^(rd) daily Patanol® dose. On Days 28-31, after        topical dose 1 on Day 28 and after the third topical dose on        days 29-31, animals were evaluated 30 minutes post Patanol®        topical dose, then challenged ˜3 minutes after evaluation, and        evaluated again 18 minutes after challenge (post topical        challenges 1, 4, 6, and 8).    -   Procedure: Mice were challenged with topical doses of 150 μg of        SRW (3 μl of 50 mg/mL) suspension in 3 μl balanced salt solution        (BSS) in each eye. Animals were randomized based on their change        from baseline hyperemia on Day 24.

a.5 Tissue Collection

Animals were euthanized and after verification of death, the right eyeand surrounding adnexa was removed and stored in Davidson's fixative for24 hours. After 24 hours of fixation, the tissue was transferred to 70%ethanol for long term storage. Eyes were paraffin embedded and 1 H&E, 1TBlue, and 1 unstained slide was made for each eye.

a.6 Statistical Methods

The data were analyzed using a two-way ANOVA with Bonferroni post-testto compare the differences of the clinical signs among groups.

B. Results

The data for hyperemia, squinting, lid swelling, and discharge are mean±SEM for N=8 eyes. The same masked observer evaluated the mice atchallenges 1, 4, 6, and 8, which occurred on Study Days 28, 29, 30, and31, respectively. For hyperemia, squinting, and discharge animals wereevaluated on a 0-4 scale of severity (with 0 being normal and 4 beingthe worst). For lid swelling, mice were graded on a scale of 0-2. Foreach endpoint, animals were evaluated 30 minutes post-dose (for Patanol®only) or just at baseline prior to challenge for that day (for all othergroups). All groups were analyzed via two-way ANOVA with Bonferronipost-test and any statistical significance versus vehicle was noted withan asterisk.

b.1 Change from Post-Dose Hyperemia—SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

Data are mean ±SEM for n=8 eyes per group. Patanol® showed astatistically lower response after the first challenge on Day 28(p<0.01). Prophylactic doses of SEQ ID NO. 112 (SYL116011) showed atrend similar to that have Patanol®, but no statistical significance wasnoted (see FIG. 26).

b.2 Change from Post-Dose Squinting—SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

Data are mean ±SEM for n=8 eyes per group (see FIG. 27). No statisticalsignificance was noted.

b.3 Change from Post-Dose Lid Swelling - SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

Data are mean ±SEM for n=8 eyes per group (see FIG. 28). No statisticalsignificance was noted.

b.4 Change from Post-Dose Discharge—SEQ ID NO. 112 (SYL116011)prophylactic versus Patanol® and Vehicle prophylactic.

Data are mean ±SEM for n=8 eyes per group (see FIG. 29). No statisticalsignificance was noted.

C. Conclusions

At the conclusion of the study, it appears as though SEQ ID NO. 112(SYL116011) prophylactic followed the same trend of reduced hyperemia,lid swelling, and discharge (see FIGS. 26-29), however is notstatistically significant when analyzing these data with two-way ANOVAwith Bonferroni post-test due to the low N used in the study.

What is remarkable is that when evaluating the effects of SEQ ID NO. 112(SYL116011) of the present invention designed to silence expression ofORAI1, the results showed a similar trend in the dose-response in theSEQ ID NO. 112 (SYL116011) group, as the group treated with Patanol®, aknown anti-allergic drug currently in the market, reducing in both casesthe allergic symptoms associated to allergic conjunctivitis in a murinemodel. It is expected that with a higher N, the analysis of the databecome statistically significant.

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What is claimed is:
 1. An siRNA molecule which targets SEQ ID NO. 1 andreduces the expression of the ORAI1 gene when introduced into a cell. 2.The siRNA molecule according to claim 1, wherein said siRNA comprises a19 nucleotide double-stranded region.
 3. The siRNA molecule according toclaim 2, wherein said siRNA is blunt-ended.
 4. The siRNA moleculeaccording to claim 1, wherein said siRNA comprises the nucleotidesequence of SEQ ID NO.
 112. 5. The siRNA molecule according to claim 1,wherein at least one nucleotide comprises a chemical modification. 6.The siRNA molecule according to claim 5, wherein said chemicalmodification is 2′-O-methylation; substitution of a uracyl ribosenucleotide with a deoxythymidine nucleotide; or a combination thereof.7. The siRNA molecule according to claim 6, wherein said chemicalmodification is on the sense strand, the antisense strand, or on boththe sense strand and the antisense strand.
 8. The siRNA moleculeaccording to claim 3, wherein said siRNA has a nucleotide sequence thatconsists of SEQ ID NO.
 112. 9. The siRNA molecule according to claim 8,wherein said siRNA has at least one nucleotide comprising a chemicalmodification.
 10. The siRNA molecule according to claim 9, wherein saidchemical modification is 2′-O-methylation; substitution of a uracylribose nucleotide with a deoxythymidine nucleotide; or a combinationthereof.
 11. The siRNA molecule according to claim 10, wherein saidchemical modification is on the sense strand, the antisense strand, oron both the sense strand and the antisense strand.
 12. The siRNAmolecule according to claim 8, wherein said siRNA has a nucleotidesequence of one of SEQ ID NO. 223 to SEQ ID NO. 229, SEQ ID NO:233, orSEQ ID NO:235.
 13. A pharmaceutical composition comprising an siRNAmolecule which targets SEQ ID NO. 1 and reduces the expression of theORAI1 gene when introduced into a cell; and a pharmaceuticallyacceptable carrier.
 14. The pharmaceutical composition of claim 13,wherein the siRNA molecule is blunt ended and comprises a 19 nucleotidedouble-stranded region.
 15. The pharmaceutical composition of claim 14,wherein said siRNA has a nucleotide sequence that consists of SEQ ID NO.112.
 16. The pharmaceutical composition of claim 15, wherein said siRNAhas at least one nucleotide comprising a chemical modification.
 17. Thepharmaceutical composition of claim 16, wherein said chemicalmodification is 2′-O-methylation; substitution of a uracyl ribosenucleotide with a deoxythymidine nucleotide; or a combination thereof.18. The pharmaceutical composition of claim 17, wherein said chemicalmodification is on the sense strand, the antisense strand, or on boththe sense strand and the antisense strand.
 19. The pharmaceuticalcomposition of claim 15, wherein said siRNA has a nucleotide sequence ofone of SEQ ID NO. 223 to SEQ ID NO. 229, SEQ ID NO:233, or SEQ IDNO:235.
 20. The pharmaceutical composition of claim 13 which isformulated for topical administration to the eye.
 21. The pharmaceuticalcomposition of claim 15 which is formulated for topical administrationto the eye.